The primary objective of this application is to better understand the electronic structure of metals in biologically important proteins. New techniques for soft-x ray spectroscopy will be develop as tools for gaining this information. As part of the technique development, new instrumentation such as high speed x-ray array detectors, better data acquisition electronics. 3He cryostats, and ultrathin window flow cells will be built. Through collaborations with theorists, new procedures for interpreting soft x-ray spectra of metalloproteins will be developed. The improved instrumentation and analysis procedures will be used to study the electron structure of Fe and Cu proteins such as cytochrome oxidase, and enzyme critical to human respiration. Nickel and cobalt, substituted for magnesium in an enzyme responsible for glucose metabolism (phosphoglucomutase), will be examine for coordination changes with pH or during crystallization. Phosphoglucomutase will be studied at eh K- edge of this natural Mg2+ component. Nickel and cobalt will also be probed in bacterial proteins such as hydrogenase and Co dehydrogenase, bote of which play important roles in the environment. Manganese, which is essential for oxygen evolution in photosynthesis, will be probed in photosystem II and smaller related protein catalase. The potential of very low energy x-ray spectroscopy from the ligand point of view, specifically nitrogen K-edges and sulfur L-edges, will be explored by examining spectral changes due to binding of metals to nitrogen or sulfur ligand s in proteins. From the interpretation of these spectra, information about the ligand field, local geometry, and orientation of the metal centers will be obtained. Orders of magnitude more flux will soon be available from undulatory and elliptical wiggler beamlines at the Advanced Light Sources, and the new equipment will allow this additional intensity to be exploited. The instrumentation, sample handling, and analysis procedures developed under this proposal will have broad applications, and will eventually be used by a significant fraction of the bioinorganic community.